There is a critical unmet need to identify the pathogenic mechanisms that drive disease progression in patients with multiple sclerosis (MS). Accumulation of axon injury and functional disability in MS are not adequately impacted by current therapies. The long-term goal of this work is to discover new strategies to prevent or reverse disease progression in MS. Despite evidence that CD8+ T cells are associated with axon injury and progression in MS, the functional role for these cells and the relevant mechanisms required for recruitment of these cells to the demyelinated brain are unknown. The antigenic targets of neuron-specific CD8+ T cells are also unknown. The overall objectives of this study are to test the mechanistic role of neuron antigen-specific CD8+ T cells in the injury of demyelinated axons and to determine whether patients with MS have such cells. The rationale is that axon injury is the primary substrate of progression in MS, axonal MHC class I expression is upregulated by inflammation and demyelination, and cytotoxic CD8+ T cells directed against neuron-specific antigens injure demyelinated axons. The central hypothesis of this proposal is that neuron antigen-specific CD8+ T cells injure demyelinated axons. Guided by strong preliminary evidence, the hypothesis will be tested using AAV-mediated transduction of neurons to drive expression of the neoantigen ovalbumin (OVA) within the context of CNS demyelination induced by cuprizone toxicity or immunization against a myelin oligodendrocyte glycoprotein-derived peptide (MOG-EAE) in hosts that have transgenic CD8+ T cells directed against the OVA- derived peptide SIINFEKL (OT-I). The study will also use autologous T cells and fibroblast-derived iPSC- derived neurons grown in microfluidic chambers to determine whether MS patient CD8+ T cells injure their own axons. Three specific aims will be pursued: 1) determine the mechanisms of CD8+ T cell-mediated axon injury in the demyelinated CNS; 2) identify the cellular locus of MHC class I expression required for axon injury and determine how demyelination drives CNS infiltration of neuron antigen-specific CD8+ T cells; 3) determine whether MS patients have neuron-antigen specific CD8+ T cells. This approach is conceptually innovative because of the proposal that demyelination and inflammation induce axonal presentation of self-peptides on MHC class I. The approach is technically innovative based on the use of novel AAV vectors to drive neoantigens in neurons within the demyelinated CNS, selective deletion of brain-resident antigen presenting cells (APCs) vs peripheral APCs, use of multiple host manipulations coupled to adoptive transfer of traceable effector cells to temporally and spatially profile anti-neuronal T cell trafficking, and the use of patient-derived neurons and autologous CD8+ T cells. This work will make a significant, powerful impact on the field by revealing the capacity of CD8+ T cells directed against neuron-specific antigens to injure demyelinated axons and by identifying such T cells in patients with MS.